XJTLU-CHINA
This year, team of XJTLU aims to simulate the process of global warming by introducing ribothermometers and chromoproteins to Escherichia coli. Global warming causes many consequences, including glacial ablation, the rise of sea level and the withering of plants. The appearance of the Earth will change as the average temperature rises: the white glaciers will melt; the green threes on the land turns into yellow and the coastal area will be flooded with sea water. To simulate this process, a genetic circuit made up with a ribothermometer that will switch at 37 degree Celsius and different color of chromoproteins was designed. Four different strains sand for different areas on earth are made: “Inland”, “Coastal”, “Marine” and “Polar”, as showed in figure 1.
Figure 1
Figure 2 shows the pathway of “Inland” E.coli. Inspired by Paris-Saclay igem team (2014), we combined a yellow chromoprotein called amilGFP (Part ID: BBa_K592010) and a blue chromoprotein called AeBlue (Part ID: BBa_K864401) to get green color in order to simulate the green color of inland area on the earth. Both chromoprotein followed with aav tail to obtain the color change distinctly from green to yellow. We introduced a tet operator to operate the expression of blue chromoprotein. The express of tetR (Part ID: BBa_C0040) is controlled by the 37℃ ribothermometer on the up steam. When the temperature is below 37℃, the tetR will not be translated and both yellow and blue chromoprotein can express, therefore, the green color shows. However, when the temperature reach above 37℃, tetR starts to be translated and bind with tetO, the gene that codes blue chromoprotein cannot be transcribed. Only the yellow chromoprotein can express. As the consequence of that, the color of the colony changed from green into yellow.
Figure 2
The repress and control system between “Coastal” and “Marine” E. coli is showed in figure 3. When the temperature is below 37℃, LuxI (Part ID: BBa_C0061) in “Marine” E. coli will not be translated, 3-oxo-hexanoyl-HSL will not express. The Lux promoter, Plux (Part ID: BBa_R0061), in “Coastal” E. coli is closed in absence of LuxR/HSL (LuxR Part ID: BBa_C0062). Therefore, tetR won’t express and both yellow and blue chromoprotein will be expressed, the color of “Coastal” E. coli is green. When the temperature rise above 37℃, the ribothermometer switches on and 3-oxo-hexanoyl-HSL starts to express, which starts the transcription of Plux and tetR. TetR binds with tet operator and stops the transcription of yellow chromoprotein. The color of “Coastal” E. coli turns into blue. This pathway simulates the process of the rise of sea level and the coastal area flooded by sea water.
Figure 3
Just like the correlation between “Marine” and “Coastal”, the color change of “Polar” E. coli is controlled by LuxR, Plux and LuxI system (Figure 4). The color of “Polar” E. coli is white (the origin color of E. coli colony) when the temperature is below 37℃ and will change into blue when the temperature is above 37℃ as the presence of 3-oxo-hexanoyl-HSL. This pathway simulates the process of the glaciers melting.
Figure 4
RNA thermometers (RNATs) are temperature-induced riboswitches that post-transcriptionally regulate gene expression in response to temperature shifts by a way of undergoing conformation changes in the secondary structure of RNA to exhibit either turn-on state or turn-off state.
Figure 1: Responsiveness of mRNA structures to environmental cues (TUDelft, 2008)
As shown in Figure 1, the hairpin structure harbors the Shine-Dalgarno sequence (SD sequence) and, in this way, make it inaccessible to the 30S unit of the bacterial ribosome, resulting in translational inactivation. (SD sequence is polypurine sequence located in around 8 nt upstream from the start codon and responsible for anchoring ribosome in the RNA single strand). Once reaching a certain temperature, hairpin structure would vanish and as a result, exposing the SD sequence to trigger the translation process.
Natural forms of RNATs were usually found in 5’ untranslated region of many eubacteria and were believed to be related to biological temperature-rising response (Nocker, 2001, cited in Neupert and Bock, 2009). Here, we would introduce two sub-families of RNATs and synthetic RNA thermometers and how they works in our project.
A conserved family of RNATs is the ROSE-like elements (ROSE stands for Repression Of heat Shock gene Expression), first discovered in the 5′ untranslated region of rhizobial heat shock genes (Nocker, 2001). All the RNAts presenting in ROSE family we known control expression of small heat shock genes (Nocker, 2001). However, most naturally occurring ROSE elements are relatively large and fold into complex secondary structures that usually contain 2-4 hairpin structures (Fig. 2). Hence, for practical reason, we tested A1 RNAT that derived from ROSE family. Thank BIT-CHINA for proving DNA sequences of this part. The possible secondary structure of A1 was simulated by RNAstructure (Fig.3). For testing results of A1 RNAT, please click here.
Figure 2: Proposed RNA secondary structure of the 5’ UTR from the prfA gene of the pathogenic bacterium Listeria monocytogenes, which belongs to ROSE family (Neupert and Bock, 2009).
Figure 3: The possible secondary structure of A1.
FourU thermometers as naturally occurring RNA thermometers, for the first time, were found in Salmonella, so called as four highly conserved uridine nucleotides are placed right opposite to the SD sequence. FourU elements usually have two hairpin structures, in the case of temperature rising, the one without SD sequence is relatively steady while the other contains hidden SD sequence would be melted. In 2008, TUDelft designed and submitted part K115002 to iGEM registry based on natural forms of FourU elements. The possible secondary structure of FourU was simulated by RNAstructure (Fig.4). We test this part this year to determine the validity of this part. For testing results of K115002, please click here.
Figure 4: The possible secondary structure of FourU.
The possibility of designing synthetic RNA thermometers was explored based on the assumption that the RNATs do function by the proposed simple hairpin-melting mechanism that expose the ribosome-binding site. Therefore, Stem-loop structure with the SD sequence embedded in the stem would be the simplest RNAT. Based on the work of Juliane Neupert, Daniel Karcher and Ralph Bock (2008), Synthetic RNAT U6 was chosen as one of our testing RNATs. The possible secondary structure of U6 was simulated by RNAstructure (Fig.5). For testing results of A1 RNAT, please click here.
Figure 5: The possible secondary structure of U6.
In the first round of experiments the RNA thermometers we’ve tested are from part registry of previous iGEM competition (K115002, K115017), support of team BIT-China (A¬1, A2, A3), literature (U9, U10) and our own design (U6-GC). We constructed parts on pET-28a as PT7-LacO- pET-28a random sequence-RNAT-eGFP. Plasmids are transformed into BL21(DE3) for expression. After transformation, samples were cultured at 30oC, 37oC and 42oC separately and induced by IPTG at respective temperature. Below are illusion of LacO-random sequence-RNAT secondary structure and their experimental results. For each structure, the 50th base is the starting point of RNAT.
1. Neupert, J. & Bock, R. (2009) 'Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria', Nature Protocols, 4 (9), August, pp.1262-1273.
2. Nocker, A. et al. mRNA-based thermosensor controls expression of rhizobial heat shock genes. Nucleic Acids Res. 29, 4800–4807 (2001).
3. TUDelft (2008) RNA thermometer [Online image]. Available from: http://2008.igem.org/File:Rna_thermometer.png (Accessed: 19th August 2015).
Chromoprotein is the protein which has conjugeted structure and can express different colors. Since our map was designed to be colorful, experiments aimed to test chromoprotein were needed. At the same time, the color can be used as reporter so that we also use chromoprotein to test other parts in our project.
There were 3 experiments related to chromoprotein. AeBlue Chromoprotein (BBa_K864401), FwYellow Chromoprotein (BBa_K1033910), amilGFP (BBa_K592010, yellow chromoprotein) and amajLime (BBa_K1033916, green-yellow chromoprotein) were four chromoproteins we used. Firstly, we tested all the chromoprotein we used. Secondly, we combined two chromoproteins together to observe their composite color for our map design. Moreover, LVA tag and AAV tag were added after chromoprotein respectively to examine their function and efficiency of degradation.
For our map design, there are two chromoproteins and one composite chromoprotein mainly for our requirement, yellow, blue and green. But due to different color of chromoprotein, the test and screen were two necessary steps to choose the fit one to show the nice color.
At the first time, AeBlue Chromoprotein (BBa_K864401) and FwYellow Chromoprotein (BBa_K1033910) were tested respectively with inducible promoters (T7 promoter). The construction is shown in Figure 1. These two parts were assembled on PET 21a plasmid and then transformed into expressible competence E.coli BL21 (DE3). The results were that the growths of bacteria were normal and the colors of these two chromoproteins were distinct. The result was shown as Figure 2 and Figure 3.
(Figure 2. The four on the left is Aeblue chromoprotein, and the four on the right is Fw Yellow chromoprotein.)
(Figure 3. These two both are Aeblue blue chromoprotein. The left one is induced under 37 degrees Celsius; the right one is induced under 16 degrees Celsius.)
Next, amilGFP yellow chromoprotein (BBa_K592010), and amajLime green-yellow chromoprotein (BBa_K1033916,) were imported in for further color in our map design. These two parts were linked after constitutive promoter (BBa_J23119) and RBS (BBa_B0034) with assembled on PSB1C3. The construction is shown in Figure 4. Then transformed into E. coli SE 2. The results were that the growths of bacteria were normal and the colors of these two chromoproteins were distinct. See results in Figure 5 and Figure 6.
(Figure 4)
(Figure5. amajLime green-yellow chromoprotein) (Figure 6. amilGFP yellow chromoprotein)
For our map design, green chromoprotein is necessary. Moreover, the changed from green to yellow (Inland) and from green to blue (Coastal) was also important to simulate global warming. Inspired by Paris-Saclay igem team (2014), we combined a yellow chromoprotein and a blue chromoprotein to get green color.
Blue and yellow chromoproteins were assembled together. Two structures were used in Figure 7. The reason why an extra promoter was added after the yellow chromoprotein was to increase the express of blue chromoprotein.
(Figure 7. The construction of expressed Aeblue and Fwyellow chromoprotein together)
The 4ml of liquid medium were used to culture the colony of BL21 (DE 3) for 5 hours, and we use IPTG to induce for 3 hours at 37 degrees centigrade. The result is shown as figure 8. However, what was unexpected was that the final colors of these two were both blue and violent instead of green. It was possible that the yellow color expressed here was not pure yellow. We plan to replace FwYellow chromoprotein.
(Figure 8. The left one is two T7 promoters and one terminator in the plasmid pet 21a. The right one is one T7 promoter and one terminator in the plasmid pet 21a)
In order to improve the color which is blue and violent instead of green, another yellow chromoprotein called amilGFP yellow chromoprotein (BBa_K592010) is brought in our test. The structure we used is shown in Figure 9.
(Figure 9. The construction of expressed Aeblue and amilGFP yellow chromoprotein together)
The 4ml of liquid medium were used to culture the colony of BL21 (DE 3) for 5 hours, and we use IPTG to induce for 12 hours at 16 degrees centigrade. The result is shown as Figure 10. As a result, what was expected was that the final color was green. These two chromoproteins are applied in our map design.
(Figure 10. Three mono-colony expressed both Aeblue and amilGFP yellow chromoprotein)
For our map design, the color change is necessary, which means the speed of degradation needs to be faster. In order to achieve this aim, we plan to add tag behind stop codon. Two tags were chosen for our testing, LVA tag and AAV tag. Due to the time limitation, only one chromoprotein, Aeblue chromoprotein was chosen to test. The construction was shown as Figure 11.
(Figure 11)
3 groups were used in our experiments: AeBlue chromoprotein, AeBlue chromoprotein with AAV tag, AeBlue chromoprotein with LVA tag. Every group has chosen 2 mono-colonies. 300ml LB was used separately culture these 6 bacteria at 37 degrees Celsius for 4 hours. Then, added 300ul IPTG to induce at 16 degrees Celsius for 12 hours. The data was collected from 5 hours to 12 hours, totally 13 points. For one point, 20ml was picked from total volume. 4ml of them was used to measure OD at 600nm and 16ml of them was used to extracted protein from strains. Firstly, 16ml bacteria were centrifuged at 6000 rpm for 5 minutes. Then discarded the supernatant and resuspended them by using 10ml PBS. Next, broke the cell of strain by using ultrasonic wave. 4ml of mixture used to centrifuge at 12000 rpm for 5 minutes. Finally, absorbance of chromoprotein was measured at 597nm by spectrophotometer.
Result:
Table 1：OD value measured at 13 time points at 600nm
Time(after induced) |
blueA |
blueB |
blueaav A |
blueaav B |
blue lva A |
blue lva B |
300min |
1.362 |
1.128 |
1.298 |
1.116 |
1.068 |
1.111 |
330min |
1.386 |
1.153 |
1.328 |
1.139 |
1.105 |
1.138 |
360min |
1.44 |
1.189 |
1.366 |
1.17 |
1.179 |
1.167 |
390min |
1.483 |
1.223 |
1.403 |
1.192 |
1.201 |
1.203 |
420min |
1.536 |
1.282 |
1.422 |
1.23 |
1.22 |
1.239 |
450min |
1.605 |
1.32 |
1.503 |
1.248 |
1.26 |
1.271 |
480min |
1.36 |
1.358 |
1.527 |
1.29 |
1.294 |
1.297 |
510min |
1.666 |
1.402 |
1.551 |
1.321 |
1.316 |
1.327 |
540min |
1.702 |
1.439 |
1.581 |
1.354 |
1.352 |
1.361 |
570min |
1.73 |
1.482 |
1.602 |
1.39 |
1.386 |
1.393 |
600min |
1.755 |
1.527 |
1.628 |
1.424 |
1.412 |
1.425 |
660min |
1.845 |
1.595 |
1.675 |
1.479 |
1.47 |
1.478 |
720min |
1.895 |
1.658 |
1.714 |
1.525 |
1.497 |
1.518 |
Table 2：Absorbance value measured at 13 time points at 597nm
Time(after induced) |
blueA |
blueB |
blueaav A |
blueaav B |
blue lva A |
blue lva B |
300min |
0.28 |
0.158 |
0.114 |
0.084 |
0.048 |
0.054 |
330min |
0.223 |
0.213 |
0.139 |
0.117 |
0.081 |
0.078 |
360min |
0.266 |
0.237 |
0.146 |
0.131 |
0.081 |
0.078 |
390min |
0.407 |
0.263 |
0.219 |
0.149 |
0.077 |
0.09 |
420min |
0.536 |
0.368 |
0.228 |
0.214 |
0.132 |
0.134 |
450min |
0.416 |
0.275 |
0.169 |
0.148 |
0.1 |
0.097 |
480min |
0.449 |
0.25 |
0.155 |
0.17 |
0.102 |
0.12 |
510min |
0.43 |
0.281 |
0.201 |
0.161 |
0.11 |
0.094 |
540min |
0.41 |
0.309 |
0.201 |
0.162 |
0.132 |
0.076 |
570min |
0.464 |
0.314 |
0.168 |
0.183 |
0.103 |
0.112 |
600min |
0.442 |
0.296 |
0.186 |
0.165 |
0.099 |
0.109 |
660min |
0.493 |
0.34 |
0.237 |
0.249 |
0.155 |
0.139 |
720min |
0.52 |
0.337 |
0.23 |
0.143 |
0.122 |
0.085 |
Table 3：Absorbance value divided by OD value
Time(after induced) |
blueA |
blueB |
Blue Average |
blueaav A |
blueaav B |
Blue AAV |
blue lva A |
blue lva B |
Blue LVA Average |
300 |
0.206 |
0.140 |
0.173 |
0.088 |
0.075 |
0.082 |
0.045 |
0.049 |
0.047 |
330 |
0.161 |
0.185 |
0.173 |
0.105 |
0.103 |
0.104 |
0.073 |
0.069 |
0.071 |
360 |
0.185 |
0.199 |
0.192 |
0.107 |
0.112 |
0.109 |
0.069 |
0.067 |
0.068 |
390 |
0.274 |
0.215 |
0.245 |
0.156 |
0.125 |
0.141 |
0.064 |
0.075 |
0.069 |
420 |
0.349 |
0.287 |
0.318 |
0.160 |
0.174 |
0.167 |
0.108 |
0.108 |
0.108 |
450 |
0.259 |
0.208 |
0.234 |
0.112 |
0.119 |
0.116 |
0.079 |
0.076 |
0.078 |
480 |
0.330 |
0.184 |
0.257 |
0.102 |
0.132 |
0.117 |
0.079 |
0.093 |
0.086 |
510 |
0.258 |
0.200 |
0.229 |
0.130 |
0.122 |
0.126 |
0.084 |
0.071 |
0.077 |
540 |
0.241 |
0.215 |
0.228 |
0.127 |
0.120 |
0.123 |
0.098 |
0.056 |
0.077 |
570 |
0.268 |
0.212 |
0.240 |
0.105 |
0.132 |
0.118 |
0.074 |
0.080 |
0.077 |
600 |
0.252 |
0.194 |
0.223 |
0.114 |
0.116 |
0.115 |
0.070 |
0.076 |
0.073 |
660 |
0.267 |
0.213 |
0.240 |
0.141 |
0.168 |
0.155 |
0.105 |
0.094 |
0.100 |
720 |
0.274 |
0.203 |
0.239 |
0.134 |
0.094 |
0.114 |
0.081 |
0.056 |
0.069 |
(Figure 12. The variation of chromoprotein after induced 300 min)
(Figure 13.)
Discussion:
As shown in the Figure 12, overview these three groups, the chromoproteins were expressed fast from nearly 300 minutes to 420 minutes. Moreover, the expressed blue chromoprotein was fluctuated in a range after 450 minutes. Compared with each chromoprotein, blue chromoprotein with lva tag has highest speed of degradation among three groups. In addition, blue chromoprotein with aav tag has higher speed of degradation than non-tag. Figure 13 shows the color of non-tag, aav tag and lva tag after induced 720 minutes. In conclusion, the tag could speed up the degradation of chromoprotein, and lva tag has higher rate of degradation than aav tag. In addition, the aav tag was chosen to use in our map in order to see clear color and high speed of degradation.
